EP1095278A2 - Differentielle diagnose von neurodegeneration - Google Patents

Differentielle diagnose von neurodegeneration

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Publication number
EP1095278A2
EP1095278A2 EP99934543A EP99934543A EP1095278A2 EP 1095278 A2 EP1095278 A2 EP 1095278A2 EP 99934543 A EP99934543 A EP 99934543A EP 99934543 A EP99934543 A EP 99934543A EP 1095278 A2 EP1095278 A2 EP 1095278A2
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EP
European Patent Office
Prior art keywords
tau
antibody
disease
antibodies
synuclein
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Granted
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EP99934543A
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English (en)
French (fr)
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EP1095278B1 (de
Inventor
Eugeen Vanmechelen
Hugo Vanderstichele
André Van de Voorde
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Fujirebio Europe NV SA
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Innogenetics NV SA
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Priority to EP99934543A priority Critical patent/EP1095278B1/de
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Publication of EP1095278B1 publication Critical patent/EP1095278B1/de
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of diagnosis of neurodegeneration.
  • the present invention relates to new methods for the differential diagnosis of neurodegeneration, making use of a combination assay detecting different neurological markers in body fluids
  • the present invention also relates to new methods for the detection of Rab3a, SNAP25 or ⁇ -synuclein in cerebrospinal fluid and to the use of these methods in a combination assay for differential diagnosis of neurodegeneration
  • Neurodegeneration is a feature of several neurological disorders Neurodegeneration may involve axonal damage, gradually evolving neuronal death, abnormalities in neurotransmitter release or receptor function, destruction of myelin, alterations in CNS blood flow, blood/brain barrier dysfunction and/or altered oxygen metabolism, difficulties in other CNS metabolic pathways and/or various other, often unknown, aspects that may cause a malfunctioning of the CNS
  • Neurodegeneration may involve axonal damage, gradually evolving neuronal death, abnormalities in neurotransmitter release or receptor function, destruction of myelin, alterations in CNS blood flow, blood/brain barrier dysfunction and/or altered oxygen metabolism, difficulties in other CNS metabolic pathways and/or various other, often unknown, aspects that may cause a malfunctioning of the CNS
  • Today different diseases have been associated with different aspects of neuronal malfunctioning (for an overview see Wilson et al .
  • Alzheimer ' s disease is the most important of all neurodegenerative diseases in which death and disappearance of nerve cells in the cerebral cortex are involved It is the most common dementia in elderly, causing distress for patients and families and economic loss in the form of the costs necessary for the long-term care of patients totally disabled by the disease
  • Frontal temporal lobe dementia is the second most common type of primary degenerative dementia and accounts for approximately 3-10% of all patients with dementia (Brun. 1993, Knopman. 1993)
  • the clinical picture is characterized by the presence of a predominating frontal lobe syndrome (Sjogren.
  • Lewy Body disease is an illness that presents with progressive dementia or psychosis Par insonian signs, which mav be absent or mild at the onset, eventually become common and rigidity is usually severe Lewy bodies are found profusely in the brainstem, basal forebrain, hypothalamic nuclei and neocortex Parkinson disease is a type of Lewy Body disease occurring in the middle or late life, with very gradual progression and a prolonged course. It can be considered as an example of neuronal system disease, involving mainly the nigrostriatal dopaminergic system. Cerebrovascular disease, on the other hand, is caused by one of several pathologic processes involving the blood vessels of the brain. It is the third leading cause of death after heart disease and cancer in developed countries and has an overall prevalence of 794 per 100 000. Five percent of the population over
  • LBD Dementia
  • Lewy Body dementia (more than 75%) are neuropathological defined as Alzheimer's disease patients while it is estimated that 15 to 25 % of the clinical diagnosed Alzheimer's disease patients have Lewy Body dementia (Hooten et al , 1998) As Lewy Body dementia is more susceptible to acetylchohnesterase treatment, differentiation of Lewy Body dementia from Alzheimer's disease is essential for optimization of treatment (Levy et al , 1994, Perry et al , 1994, Wilcock et al ,
  • frontal temporal lobe dementia is often misdiagnosed as other types of dementia or other psychiatric disorders since the symptoms of frontal temporal lobe dementia can also be observed in other disorders
  • CNS central nervous system
  • microtubule-associated protein tau for example, is a major protein component of paired helical filaments (PHF) and neurofibrillar tangles (NFT) (Brion et al , 1985, Delacourte and Defossez, 1986, Grundke-Iqbal et al , 1986, Kosik et al , 1986, Wood et al , 1986, Kondo et al ,
  • PHF paired helical filaments
  • NFT neurofibrillar tangles
  • Tau protein exists in different isoforms, of which 4 to 6 are found in adult brain but only
  • 1 isoform is detected in fetal brain
  • the diversity of the isoforms is generated from a single gene on human chromosome 17 by alternative mRNA splicing (Himmler, 1989, Goedert et al , 1989,
  • tau protein As deduced from molecular cloning, is a stretch of 31 or 32 amino acids, occurring in the carboxy-terminal part of the molecule, which can be repeated either 3 or 4 times Additional diversity is generated through 29 or 58 amino acid -long insertions in the NH 2 -terminal part of tau molecules (Goedert et al , 1989)
  • In vivo tau promotes microtubule assembly and stability in the axonal compartment of neurons by interactions involving its microtubule binding domain which is localized in the repeat region of tau (255-381) (Lewis et al , 1988) In normal circumstances adult brain contains 2 - 3 mole phosphate per mole of tau (Selden and Pollard, 1983, Ksiezak-Reding et al , 1992)
  • NSE neuron-specific enolase
  • NSE represent 3% of total soluble brain protein
  • Nara et al (1988) showed that a high NSE level in cerebrospinal fluid or serum is correlated with poor outcome and death in comatose children.
  • increased serum NSE is not necessarily of CNS origin.
  • tissues including peripheral neurons, endocrine glands, lymphocytes, red blood cells, and platelets contain
  • ⁇ -amyloid a 40-43 amino acids long peptide, is derived via proteolytic cleavage from a large precursor protein, called amyloid precursor protein or APP. Amyloid is produced during metabolism of normal cells. The amyloid peptide exhibits a high degree of heterogeneity. Two major forms of ⁇ -amyloid have been identified, ⁇ -amyloid ( ⁇ -40 ) and ⁇ -amyloid ( ⁇ - 42) .
  • ⁇ -amyloid d ⁇ is a major constituent of the neuritic plaques of Alzheimer's disease, Down's syndrome and normal aged brains.
  • GAP-43 Growth associated protein-43
  • B-50 is a nervous tissue specific protein, primarily localized to the axons and presynaptic terminals. GAP-43 is considered to play a major role in neuronal growth, neurite formation, and in regeneration and neuronal sprouting (Skene and Willard, 1981; Basi, 1987; Benowitz et al., 1989; Mercken et al., 1992a).
  • Synapse proteins have different roles in synapse function. Proteins such as synapsin are important in determining the amount of vesicles available for fusion, while Rab3a and rabphilin are important in targeting the vesicles to the membrane.
  • the docking process is determined by a molecular complex of synaptobrevin, SNAP25, Sec and syntaxin, while it is believed that CSP and synaptotagmin play an important role in the Ca 2+ -dependent release of the content of the vesicle.
  • Alpha-synuclein is abundant in synapses of the substantia nigra and basal ganglia, and belongs to a family of proteins including -synuclein and ⁇ -synuclein.
  • Alzheimer's disease is characterized by abundant extracellular senile plaques, intracellular tangles, and synapse loss.
  • Tau and ⁇ -amyloid ( ⁇ . 42 ) are essential components of respectively these tangles and plaques, the two diagnostic structures in the neuropathological examination of AD.
  • tau and ⁇ -amyloid ( i -42) have been detected in cerebrospinal fluid (CSF) and it is now well-established that CSF-tau and CSF- ⁇ -amyloid(i - 2) can be used as neurological markers for Alzheimer's disease, although it is not yet known how changes in CSF levels relate to the pathophysiology of Alzheimer's disease.
  • CSF-tau is increased in Alzheimer patients compared to age-matched controls and relates to the number of tangles in the brain, while ⁇ -amyloid ( ⁇ -42) is reduced in Alzheimer's disease.
  • ⁇ -amyloid ( ⁇ . 42) is probably not related to plaque formation as it is found reduced in dementia without senile, diffuse plaques such as frontal lobe dementia.
  • studies on brain tissue suggest a relationship for plaques and certainly tangles to the degree of dementia, the levels of CSF-tau and CSF- ⁇ -amyloid d ⁇ are not consistently related to the degree of dementia, as defined by the Mini-Mental State, and overlap with other types of dementia occurs as well.
  • ⁇ -amyloid(i- 4 o) as a neurological marker in addition to tau and ⁇ -amyloid(i - 2) (Shoji et al., 1998) is not improving the diagnostic assay for Alzheimer's disease as the level of ⁇ -amyloid ( ⁇ -4 o) in the Alzheimer's disease patients does not change compared to the normal controls (Motter et al., 1995).
  • GAP-43 Studies on brain GAP-43 have suggested decreased levels in the frontal cortex in Alzheimer's disease but increased levels in other regions (Coleman et al., 1992). No study has been performed on GAP-43 in body fluids of patients with dementia disorders.
  • synapse loss Another important structural alteration in brains of AD patients is synapse loss.
  • synapse loss is the major correlate of the degree of dementia (Terry et al., 1991).
  • synaptophysin immunoreactivity was observed.
  • a similar reduction has been documented for other synapse proteins: synaptotagmin, Rab3a, synaptobrevin and syntaxin (Blennow et al., 1996; Davidsson et al., 1996; Shimohama et al., 1997; Ferrer et al., 1998).
  • synapse proteins play a pathological role.
  • Two mutations in ⁇ -synuclein were detected in two rare forms of Familial Parkinson disease and ⁇ -synuclein was characterized as a major component in lewy bodies. Lewy body formation in vivo may result from synuclein accumulation, which may be the consequence of a reduction in the fast axonal transport or overexpression of synuclein (Jensen et al, 1998).
  • Chromogranin has already been used as a marker for synapse loss in CSF, but a reduction was only shown in 'pure' or type I Alzheimer's disease (Blennow et al., 1995). Shortly thereafter synaptotagmin I was shown to be present in CSF (Davidsson et al., 1996). In this study it was demonstrated that synaptotagmin is selectively reduced in the left hippocampal formation and
  • the aim of the present invention is to provide methods for specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual.
  • Alzheimer's disease versus Parkinson disease Alzheimer's disease versus Parkinson disease.
  • Alzheimer's disease versus Lewy Body dementia Alzheimer's disease versus Lewy Body dementia.
  • It is another aim of the present invention is to provide a method for the diagnosis of neurodegeneration resulting from perinatal asphyxia.
  • the present invention relates to methods for specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual. These methods involve the determination of the level of at least three neurological markers in one or more body fluid samples of said individual, whereby the type and degree of neurodegeneration is reflected by a quantitative change in the level of all of said neurological markers compared to a control sample.
  • neurodegeneration and neurodegenerative condition used in the present application stand for the same and are used interchangeable throughout the application. These terms include any condition of the brain that is associated with a neuronal malfunctioning.
  • diseases associated with neurodegeneration are cited in Wilson et al. (1991). They include Alzheimer's disease, stroke (Focal brain injury), diffuse brain injury, vascular disease, Parkinson disease, Lewy Body Disease, Creutzfeld Jacob Disease, Frontal temporal lobe dementia, Guilain Barre Syndrome, Multiple Sclerosis, Normal Pressure Hydrocephalus, Amyotrophic Lateral Sclerosis, Schizophrenia, Depression, Neurolathyrisme, Epilepsy and Asphyxia.
  • stroke Fecal brain injury
  • diffuse brain injury vascular disease
  • Parkinson disease Lewy Body Disease
  • Creutzfeld Jacob Disease Frontal temporal lobe dementia
  • Guilain Barre Syndrome Guilain Barre Syndrome
  • Multiple Sclerosis Normal Pressure Hydrocephalus
  • Amyotrophic Lateral Sclerosis Schizophrenia
  • Depression Neurolathyrisme
  • Neurodegeneration also includes any kind of brain damage or any condition of the brain that is associated with a neuronal malfunctioning and which is caused by a specific inducing agent.
  • the neurodegenerative condition to be specifically detected, quantified and/or differentially diagnosed is chosen from the group consisting of Alzheimer's disease, Lewy Body Disease, Parkinson disease and frontal temporal lobe dementia.
  • Lewy Body Disease is used for any disease showing lewy bodies in the brainstem, basal forebrain, hypothalamic nuclei and/or neocortex.
  • Lewy Body Disease includes Parkinson disease, multiple system atrophy and Lewy Body dementia.
  • the neurodegenerative condition to be specifically detected, quantified and/or differentially diagnosed is induced by hypoxic-ischemic events, chemotherapy, radiotherapy, or by exposure to chemical compounds or irradiation. More particularly, neurodegeneration can be induced by chemotherapy or radiotherapy during the treatment of leukemia or brain tumor.
  • specific detection of neurodegeneration means that a higher sensitivity and specificity is obtained for the association of a certain disease or a certain cause of neurological disorder with a certain neurodegenerative condition than would be obtained when less than three neurological markers were used for diagnosis.
  • Quantification of neurodegeneration means that the degree of neuronal malfunctioning due to a certain neurodegenerative condition is determined.
  • the expression "differential diagnosis of neurodegeneration” as used in the present invention refers to the discrimination between various neurodegenerative conditions in this way that a certain disease or a certain cause of neurological disorder is associated with a certain neurodegenerative condition.
  • the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual is accomplished by the detection of at least three different neurological markers in one or more body fluid samples of said individual, making use of an immuno-assay comprising the steps of:
  • the process for the detection of the immunological binding can then be carried out by bringing together said antigen-antibody complex formed by the antigen and the antibody recognizing one of the neurological markers with: a) a secondary antibody (or detector antibody)
  • the antibodies used in the invention are in an immobilized state on a suitable support.
  • the antibodies may be present on up to three (or more in case more than 3 neurological markers are detected) different supports or on the same support.
  • the immunological binding of each of them may be detected by a specific marker.
  • the antibodies may be present on distinct locations of the same support. In the latter case, detection may occur with a general marker that detects the immunological binding of any of these antibodies.
  • the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.
  • the present process may be put into practice by using any other immunoassay format known to the person skilled in the art.
  • epitope refers to that portion of the antigen-antibody complex that is specifically bound by an antibody-combining site. Epitopes may be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known in the art.
  • body fluids refers to all fluids that are present in the human body including but not limited to blood, lymph, urine and cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the present invention relates to a method as described above wherein the body fluid sample is chosen from the group consisting of a cerebrospinal fluid sample and a blood sample.
  • the blood sample can include the whole sample as taken from the patient. More preferably the blood sample includes a plasma sample or a serum sample.
  • the methods of the present invention it is also possible to detect the same marker in two different body fluids (in combination with the detection of at least one other marker) or to detect the same marker in three different body fluids.
  • two neurological markers are detected in cerebrospinal fluid and one of these neurological markers is also detected in plasma.
  • the neurological markers that are detected in the method of the present invention can be any protein associated with certain types of neuronal cells or cell function, of which the level in one or more body fluids under conditions of neurodegeneration is indicative for the disease process or the cause of neurological disorder. Some neurological markers are elevated and others are reduced in one or more body fluids under a certain neurological condition. Any possible combination of 3, 4, 5, 6, 7, 8 or more neurological markers that have an altered level in a certain body fluid under a certain neurological condition can be used for the specific detection, quantification and/or differential diagnosis of said neurological condition in an individual.
  • Possible neurological markers used for specific detection, quantification and/or differential diagnosis of neurodegeneration include: tau, neuron-specific enolase (NSE), beta-amyloid ( i. 42) , beta-amyloid(i -40 ), neuromodulin, synapse proteins (such as Rab3a, SNAP25, ⁇ -synuclein, synapsin, synaptotagmin, synaptobrevin, syntaxin, rabphilin, n-sec, cystein string protein and others), glial fibrillary acidic protein (GFAP), SI 00, LL6, TNF, IL1, LL2, neurofilament (NF), myelin basic protein (MBP) and 14-3-3.
  • GFAP glial fibrillary acidic protein
  • SI 00 LL6, TNF, IL1, LL2, neurofilament (NF), myelin basic protein (MBP) and 14-3-3.
  • MBP myelin basic protein
  • Antibodies specifically recognizing tau include Alz50 (Ghanbari et al, 1990), Ab423 (Harrington et al., 1991), AT8 (International application published under
  • Antibodies that specifically recognize NSE include 10C1 and 2E7 and others from Innogenetics (Gent, Belgium), commercially available antibodies such as can be obtained from Dako (Glostrup, Denmark; Cat No BBS/NC/VI-H14), from Biogenex (San Ramon, CA, USA; Cat Nos MA055-5C and AM055-5M), from RDI (Flanders, NJ, USA; Cat No RDI-TRK4N6), from Roche Diagnostic Systems (Basel, Switzerland; Cat No 07 34373), from Immunosource (Brussels, Belgium; Cat Nos CLA 73/5 and CR7041M) and from Cortex Biochem (San Leandro, CA, USA; Cat No CR7047).
  • Antibodies that specifically recognize ⁇ -amyloid include 2H3, 8E5 (Johnson- Wood et al., 1997), 10H3 (Majocha et al., 1992; Friedland et al., 1994), 2G3 (Citron et al., 1996), BA-27 and BC-05 (Suzuki et al., 1994), BNT77 (Asami-Odaka et al., 1995), 369.2B (K ⁇ nig et al., 1996), 22C11 (Lannfelt et al, 1995), 6E10 (Kim et al,, 1990) and AMY-33 (Stern et al, 1990).
  • Antibodies that specifically recognize neuromodulin include NM2 (Oestreicher et al., 1994), NM4 (Six et al, 1992), NM1, NM3, NM6, NM7 and NM8 (Mercken et al, 1992a). But any other antibody known in the art that specifically recognizes neuromodulin can be used as well.
  • Antibodies specifically recognizing Rab3a include commercially available antibodies such as can be obtained from Transduction Labs (Lexington, KY, USA; Cat No R35520). Also other antibodies commercially available or described in the art that recognize Rab3a can be used.
  • Antibodies specifically recognizing SNAP25 include commercially available antibodies such as can be obtained from Serotec (Oxford, UK; Cat No SP12), from Sternberger Monoclonals Inc. (Distributed by Affinity Research Products Lim., Mamhead, Wales, UK; Cat No SMI-81), from Chemicon (Temecula, CA, USA; Cat No MAB331) and from Transduction Labs (Lexington, KY, USA; Cat No S35020). This list of antibodies recognizing SNAP25 is not complete and other antibodies commercially available or described in the art that recognize SNAP25 can be used as well.
  • Antibodies specifically recognizing ⁇ -synuclein include commercially available antibodies such as can be obtained from Transduction Labs (Lexington, KY, USA; Cat No S63320). Also other antibodies commercially available or described in the art that recognize ⁇ -synuclein can be used Also for the specific detection of other synapse proteins that can possibly be used as neurological markers, various antibodies are commercially available and/or known in the art Antibodies specifically recognizing SI 00 include commercially available antibodies such as can be obtained from Biogenex (San Ramon, CA, USA, Cat Nos MA058-C and AM058-5M) and from
  • Antibodies specifically recognizing 14-3-3 include commercially available antibodies such as can be obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
  • Antibodies specifically recognizing neurofilament include commercially available antibodies such as can be obtained from Innogenetics (Gent, Belgium, Cat Nos M-011 and M-005) and from Alexis
  • fragments derived from these monoclonal antibodies such as Fab, F(ab)' 2 , ssFv ("single chain variable fragment") and other antibody like constructs that retain the variable region of the antibody, providing they have retained the original binding properties, can be used in a method of the present invention
  • Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases
  • monoclonal antibodies, or fragments thereof can be modified for various uses
  • miniantibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptabodies can be used in a method of the invention The preparation and use of these fragments and multivalent antibodies has been described extensively in International Patent Application WO 98/29442
  • the monoclonal antibodies used in a method of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for
  • the monoclonal antibodies used in a method of the invention may be human monoclonal antibodies
  • the term "humanized antibody” means that at least a portion of the framework regions of an i munoglobulin is derived from human immunoglobulin sequences
  • the antibodies used in a method of the present invention may be labeled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
  • At least one of the neurological markers to be detected in a method as described above is chosen from the group consisting of: tau, phospho- tau, ⁇ -amyloid(i -42 ), ⁇ -amyloid -4 o), neuromodulin, neuron-specific enolase and/or synapse proteins.
  • tau phospho- tau
  • ⁇ -amyloid -4 o neuromodulin
  • neuron-specific enolase neuron-specific enolase
  • synapse proteins any possible combination of 3, 4, 5, 6, 7, 8 or more markers of which one is chosen from the above group can be used for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual. It is clear that also more than one (i.e. 2, 3,
  • one, more preferably two, most preferably three of the neurological markers to be detected in a method as described above are chosen from the following groups:
  • tau is detected in one body fluid, preferably CSF and ⁇ -amyloid ( ⁇ - ) is detected in 2 different body fluids, preferably CSF and plasma.
  • At least one of the neurological markers to be detected in a method as described above is a synapse protein chosen from the group consisting of Rab3a, SNAP25 and ⁇ -synuclein. Any possible combination of 3, 4, 5, 6, 7, 8 or more markers of which one is chosen from the above group of synapse proteins can be used for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual.
  • the present invention also relates to a new method for the detection of Rab3a in cerebrospinal fluid, comprising at least the following steps:
  • a preferred monoclonal antibody for use in the method of the invention can be obtained from Transduction Labs (Lexington, KY, USA; Cat No R35520).
  • the monoclonal antibody used in the invention is in an immobilized state on a suitable support.
  • the present process may be put into practice by using any other immunoassay format known to the person skilled in the art.
  • the process for the detection of the immunological binding can then be carried out by bringing together said antigen-antibody complex formed by the antigen and the antibody recognizing
  • Rab3a with: a) a secondary antibody (or detector antibody)
  • a marker either for specific tagging or coupling with said secondary antibody, with said marker being any possible marker known to the person skilled in the art; c) appropriate buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the neurological marker-primary antibody complex and/or between the bound second antibody and the marker; and d) possibly also, for standardization purposes, purified proteins or synthetic peptides reactive with the antibodies that recognize Rab3a.
  • a polyclonal Rab3a serum may be used as a detector antibody.
  • the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.
  • the present invention also relates to a new method for the detection of SNAP25 in cerebrospinal fluid comprising at least the following steps: - obtaining a cerebrospinal fluid sample from an individual; and
  • a preferred monoclonal antibody for use in the method of the invention can be obtained from Serotec (Oxford, UK; Cat No SP12), from Sternberger Monoclonals Inc. (Distributed by Affinity Research Products Lim., Mamhead, Wales, UK; Cat No SMI-81), from Chemicon (Temecula, CA, USA; Cat No MAB331) or from Transduction Labs (Lexington, KY, USA; Cat No S35020).
  • the monoclonal antibody used in the invention is in an immobilized state on a suitable support.
  • the present process may be put into practice by using any other immunoassay format known to the person skilled in the art.
  • the process for the detection of the immunological binding can then be carried out by bringing together said antigen-antibody complex formed by the antigen and the antibody recognizing SNAP25 with: a) a secondary antibody (or detector antibody)
  • a marker either for specific tagging or coupling with said secondary antibody, with said marker being any possible marker known to the person skilled in the art; c) appropriate buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the neurological marker-primary antibody complex, and/or the bound secondary antibody and the marker; and d) possibly also, for standardization purposes, purified proteins or synthetic peptides reactive with the antibodies that recognize SNAP25.
  • a polyclonal SNAP25 serum may be used as a detector antibody.
  • the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.
  • the present invention also relates to a new method for the detection of ⁇ -synuclein in cerebrospinal fluid comprising at least the following steps:
  • any antibody that allows specific detection of ⁇ -synuclein in cerebrospinal fluid can be used in this new method.
  • a preferred monoclonal antibody for use in the method of the invention can be obtained from Transduction Labs (Lexington, KY, USA; Cat No R35520).
  • the monoclonal antibody used in the invention is in an immobilized state on a suitable support. Possibly this immobilized state can be a microtiter plate, coated or not coated with anti-IgG.
  • the present process may be put into practice by using any other immunoassay format known to the person skilled in the art.
  • the process for the detection of the immunological binding can then be carried out by bringing together said antigen-antibody complex formed by the antigen and the antibody recognizing ⁇ -synuclein with: a) a secondary antibody (or detector antibody)
  • a marker either for specific tagging or coupling with said secondary antibody, with said marker being any possible marker known to the person skilled in the art; c) appropriate buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the neurological marker-primary antibody complex, and/or the bound secondary antibody and the marker; and d) possibly also, for standardization purposes, purified proteins or synthetic peptides reactive with the antibodies that recognize ⁇ -synuclein.
  • the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.
  • these methods for the detection of Rab3a, SNAP25 and/or ⁇ - synuclein can be used in combination with a method for detection of one or more other neurological markers in order to specifically detect, quantify and/or differential diagnose neurodegeneration in an individual.
  • these methods for the detection of Rab3a, SNAP25 and/or ⁇ -synuclein can be used in combination with a method for detection of one or more neurological markers chosen from the group consisting of tau, phospho-tau, ⁇ -amyloid ( ⁇ . 42) , ⁇ -amyloid(i- o ) , neuromodulin, neuron-specific enolase (NSE).
  • one or more neurological markers chosen from the group consisting of tau, phospho-tau, ⁇ -amyloid ( ⁇ . 42) , ⁇ -amyloid(i- o ) , neuromodulin, neuron-specific enolase (NSE).
  • the neurological markers used for the specific detection, quantification and/or differential diagnosis of neurodegeneration can be chosen from one of the following groups:
  • -tau phospho-tau, NSE, ⁇ -amyloid - 2 ), ⁇ -amyloid( 1-4 o), neuromodulin or Rab3a
  • -tau phospho-tau, NSE, ⁇ -amyloid ( ⁇ - 2 ), ⁇ -amyloid(i. 0 ), neuromodulin or SNAP25
  • -tau phospho-tau, NSE, ⁇ -amyloid (1-4 ), ⁇ -amyloid(i- o,, neuromodulin or ⁇ -synuclein.
  • Any possible combination of 3, 4, 5, 6 or 7 markers from the above groups can be used for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual.
  • the methods for the detection of Rab3a, SNAP25 and/or ⁇ -synuclein can be used together for the specific detection, quantification and/or differential diagnosis of neurodegeneration. Accordingly the present invention relates to a method wherein two or three of the neurological markers are chosen from the group consisting of Rab3a, SNAP25 and ⁇ - synuclein.
  • a very specific embodiment relates to a method as described above for the specific detection or quantification of Alzheimer's disease and or Lewy Body Disease and/or for the differential diagnosis of Alzheimer's disease versus Lewy Body Disease, wherein:
  • ⁇ -synuclein is determined in a cerebrospinal fluid sample
  • Another very specific embodiment relates to a method for the specific detection or quantification of Alzheimer's disease and/or for the differential diagnosis of Alzheimer's disease versus other dementia wherein:
  • the level of tau, ⁇ -amyloid ( ⁇ - 2) and Rab3a is determined in a cerebrospinal fluid sample;
  • Another very specific embodiment relates to a method for the specific detection or quantification of Alzheimer's disease and/or Parkinson's disease and/or for the differential diagnosis of Alzheimer's disease versus Parkinson's disease wherein the level of tau, ⁇ - amyloid(i -42 ) and neuromodulin is determined in a cerebrospinal fluid sample.
  • Another very specific embodiment relates to a method for the specific detection or quantification of neurodegeneration induced by chemotherapy, exposure to chemical compounds and/or irradiation wherein the level of tau, neuron- specific enolase and neuromodulin is determined in a cerebrospinal fluid sample.
  • Another very specific embodiment relates to a method for the specific detection or quantification of neurodegeneration induced by chemotherapy, exposure to chemical compounds and/or irradiation in an individual treated for leukemia or brain tumor wherein the level of tau, neuron-specific enolase and neuromodulin is determined in a cerebrospinal fluid sample.
  • Another very specific embodiment relates to a method for the specific detection or quantification of neurodegeneration induced by prenatal asphyxia wherein at least three neurological markers are detected.
  • Another very specific embodiment relates to a method for the specific detection or quantification of Frontal Temporal Lobe dementia and/or for the differential diagnosis of
  • Frontal Temporal Lobe dementia versus other dementia wherein the level of tau, phospho-tau and ⁇ -amyloid ( i - 2 ) is determined in a cerebrospinal fluid sample.
  • Another very specific embodiment relates to a method for the specific detection or quantification of vascular problems in Alzheimer's disease, for the differential diagnosis of different forms of Alzheimer's disease and/or for the differential diagnosis of Alzheimer's disease versus other dementia, wherein at least the level of:
  • ⁇ - tau and ⁇ -amyloid(i - 2) is determined quantitatively in a cerebrospinal fluid sample and the level of ⁇ -amyloid ( ⁇ - 2) is determined quantitatively in a plasma sample; or
  • -phospho-tau and ⁇ -amyloid ( ⁇ -42 ) is determined quantitatively in a cerebrospinal fluid sample and the level of ⁇ -amyloid(i. 2) is determined quantitatively in a plasma sample; or
  • - tau and phospho-tau is determined quantitatively in a cerebrospinal fluid sample and the level of ⁇ -amyloid ( ⁇ . 42 ) is determined quantitatively in a plasma sample; or
  • -tau, phospho-tau and ⁇ -amyloid(M 2 ) is determined quantitatively in a cerebrospinal fluid sample.
  • the above methods for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual by determination of level of at least three neurological markers in body fluids of said individual can be used alone, in combination with easily monitored neurological endpoints (e.g. leucocyte count) or in combination with the measurement of drug concentrations in plasma.
  • the present invention also relates to a diagnostic kit for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual, comprising at least three antibodies each recognizing a different neurological marker in one or more body fluid samples of said individual. More particularly, the present invention relates to a diagnostic kit for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual, comprising at least a support such as a microtiterplate with, together or in separate wells, at least three antibodies each recognizing a different neurological marker in one or more body fluid samples of said individual.
  • the present invention also relates to a kit for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual, comprising:
  • a support such as a microtiterplate comprising, together or in separate wells, at least three antibodies (primary antibodies or capturing antibodies) each recognizing a different neurological marker,
  • * which can be a monoclonal antibody being capable of forming an immunological complex with an epitope of the neurological marker-primary antibody complex but not with the primary antibody alone;
  • * which can be a polyclonal antibody being capable of forming an immunological complex with epitopes of the neurological marker-primary antibody complex but not with the primary antibody alone, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized neurological marker or neurological marker-primary antibody complex;
  • the present invention relates to diagnostic kits as described above each designed for performing one or more of the methods as described above. More particularly the present invention relates to a diagnostic kit as described above comprising at least antibodies that specifically recognize: - ⁇ -synuclein; or
  • the present invention also relates to a kit for the detection of Rab3a in cerebrospinal fluid, comprising at least a monoclonal antibody recognizing Rab3a.
  • the present invention also relates to a kit for the detection of Rab3a in cerebrospinal fluid, comprising at least a support such as a microtiterplate comprising a monoclonal antibody recognizing Rab3a.
  • the present invention relates to a kit for the detection of Rab3a in cerebrospinal fluid, comprising:
  • a support such as a microtiterplate comprising a monoclonal antibody recognizing Rab3a (primary antibody or capturing antibody);
  • * which can be a polyclonal antibody being capable of forming an immunological complex with an epitope of the Rab3a-primary antibody complex but not with the primary antibody alone, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized Rab3a or Rab3a- primary antibody complex;
  • the present invention also relates to a kit for the detection of SNAP25 in cerebrospinal fluid, comprising at least a monoclonal antibody recognizing SNAP25.
  • the present invention also relates to a kit for the detection of SNAP25 in cerebrospinal fluid, comprising at least a support such as a microtiterplate comprising a monoclonal antibody recognizing SNAP25.
  • the present invention relates to a kit for the detection of SNAP25 in cerebrospinal fluid, comprising:
  • a support such as a microtiterplate comprising a monoclonal antibody recognizing SNAP25 (primary antibody or capturing antibody);
  • the present invention also relates to a kit for the detection of ⁇ -synuclein in cerebrospinal fluid, comprising at least a monoclonal antibody recognizing ⁇ -synuclein.
  • the present invention also relates to a kit for the detection of ⁇ -synuclein in cerebrospinal fluid, comprising at least a support such as a microtiterplate comprising a monoclonal antibody recognizing ⁇ -synuclein.
  • the present invention relates to a kit for the detection of ⁇ -synuclein in cerebrospinal fluid, comprising:
  • a support such as a microtiterplate comprising a monoclonal antibody recognizing ⁇ -synuclein (primary antibody or capturing antibody) directly linked to the microtiterplate, possibly by an anti-IgG antibody;
  • * which can be a monoclonal antibody being capable of forming an immunological complex with an epitope of the ⁇ -synuclein-primary antibody complex but not with the primary antibody alone, or
  • * which can be a polyclonal antibody being capable of forming an immunological complex with an epitope of the ⁇ -synuclein-primary antibody complex but not with the primary antibody alone, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized ⁇ -synuclein or ⁇ - synuclein-primary antibody complex;
  • the present invention also relates to the use of any method or any kit as described above for therapeutic monitoring and/or determination of the effectiveness of a certain treatment.
  • the content of all references describing antibodies specific for any of the disclosed markers is hereby incorporated by reference into the description of the present invention.
  • the following examples merely serve to illustrate the present invention.
  • AD Alzheimer's disease
  • PD Parkinson disease
  • CJD Creutzfeld Jacob Disease
  • GBS Guilain Barre Syndrome
  • ALS ALS
  • Amyotrophic Lateral Sclerose MS: Multiple Sclerose; P-tau: phospho-tau; NSE: neuron-specific enolase; BA(l-42): ⁇ -amyloid ()-42) ; BA(1-
  • AU Arbritary units.
  • Type Subtype Specification Total (longitudinally) Age (range) M/F
  • Type Subtype Specification Total (longitudinally) Age (range) M/F
  • COPADM 1 and COP ADM 2 Prednisolone 60 mg/m2/d PO day 1 - 5 45
  • IT intra thecal
  • IV intravenous
  • PO per os
  • LP lumbal puncture
  • MTX methotrexate
  • Ara-C arabinoside
  • Table 7b Treatment Protocol for patients with non-B-cell ALL/NHL (EORTC 58881).
  • Protocol I induction Prednisolone 60 mg/m2/d PO day 8 - 28
  • Protocol I consolidation Cyclophosphamide 1000 mg/m2 IV day 36, 63
  • Protocol II induction Dexamethasone 6 mg/m2/d PO day 1 - 21 (35 days) 3 mg/m2/d PO day 22 - 35
  • Protocol II consolidation Cyclophosphamide 1000 mg/m2 IV day 36 (14 days) 6-thioguanine 60 mg/m2 PO day 36 - 49
  • Table 7c Treatment Protocol for patients with AML (EORTC 58921).
  • IT intra thecal
  • IV intravenous
  • PO per os
  • SC subcutanous
  • LP lumbal puncture
  • Ara-C arabinoside
  • VP16
  • Table 8 Average cerebrospinal fluid levels of tau, ⁇ -amyloid ( ⁇ . 2 ) and neuromodulin for 4 groups of patients as described in example 7.
  • Control 70 36/34 45 ⁇ 16 120 ⁇ 78 466 ⁇ 168 504 ⁇ 283
  • Table 9 Average cerebrospinal fluid levels of tau, ⁇ -amyloid 1 _ 2 ) and neuromodulin in patients with Alzheimer's disease, Parkinson disease and in age-mached controls.
  • NM neuromodulin
  • AD Alzheimer's Disease
  • PD Parkinson Disease
  • C control patients.
  • FIG. 1 Western blot as described in example 1 3, showing Rab3a immunoreactivity in temporal cortex of Alzheimer's disease and control brains 1 12 8 ⁇ l Control patient n°3, 2 6 4 ⁇ l Control patient n°3, 3 3 2 ⁇ l Control patient n°3, 4 1 6 ⁇ l Control patient n°3, 5 1 0 ⁇ l Control patient n°3, 6 10 ⁇ l Control patient n°3, 7 10 ⁇ l AD patient n°l, 8 10 ⁇ l AD patient n°2, 9 10 ⁇ l Control patient n°l, 10 10 ⁇ l Control patient n°2, 11 10 ⁇ l AD patient n°3, 12 10 ⁇ l Control patient n°4
  • FIG. 1 Stability of synapse proteins Rab3a (a) and SNAP25 (b) in cerebrospinal fluid
  • the degradation of synapse proteins was quantified via a sandwich ELIS A specific for the synapse protein as described in example 1 5
  • Purified synapse proteins were spiked in a pool of CSF and incubated overnight at 37°C (legend Rab3a in CSF, SNAP25 in CSF)
  • As a control the synapse protein was spiked in the same pool of CSF and directly quantified (legend Rab3a, SNAP25)
  • the stability was also assayed in 1% BSA (results not shown)
  • FIG. 3 Demonstration of the specificity of the antibody from Transduction Labs (Lexington, KY, USA, Cat No S63320) for ⁇ -synuclein A Coumassie stained gel, B Western blot developed with an anti-His monoclonal antibody to reveal expression of all products, C Western blot developed with the monoclonal antibody from Transduction Labs (Lexington, KY, USA) Lane 1 E. coli expressing ⁇ -synuclein, lane 2 E. coli expressing ⁇ -synuclein, lane 3 E. coli expressing ⁇ -synuclein, lane 4 control lane with an E. coli expressing neuron-specific enolase, lane 5 molecular weight standards, lane 6 E. coli strain without any plasmid
  • FIG. 9 Tau values at diagnosis, before any treatment was given 1 AML (3), 2 AML-CNS+ (1), 3 Down AML (2), 4 Myelodysplasia (2), 5 Others [(Medulloblastoma(2), rhabdomyosarcoma (2), intracranial germinoma (1)], 6 B-NHL (8), 7 Hodgkin's Disease (3), 8 Down NB ALL (1), 9 NB ALL (21), 10 NB ALL- Brachman syndrome (1), 11 NB ALL- CNS + (1), 12 NB ALL-VHR (4), 13 Controls (6) (number of patients)
  • FIG. 10 Concentrations of the CSF neurological markers tau (a), neuromodulin (b), ⁇ - amyloid (1 . 42 ) (c) and NSE (d), serum LDH (e) and CSF WBC (f) at day 1 1 AML, 2 AML - CNS+, 3 Down AML/MDS, 4 Myelodysplasia, 5 Chronic myelomic leukemia, 6 B-NHL, 7 Hodgkin's Disease, 8 Down NB ALL, 9 NB ALL, 10 NB ALL - Brachman syndrome, 11 NB ALL - CNS+, 12 NB ALL - VHR, 13 LCH, rhabdomyosarcoma, germinoma, medulloblastoma, choriocarcinoma, 14 Controls, retinoblastoma (healthy), hemofagocytose (gezond HLH) Detection methods for the markers are described in example 3
  • NHL patients The number on the X-axis corresponds to the LP number as given in table 7a
  • FIG. 15 Individual levels of tau (a), ⁇ -amyloid ( ⁇ - 2 ) (b) and neuromodulin (or Growth- Associated Protein 43) (c) in individuals classified as neurological controls (Guilain-Barre Syndrome, Multiple Sclerosis, etc) (1 CONT), people with memory impairment (2 MEM), a presymptomatic Familial Alzheimer patient (PS1 mutation) (3 PFAD), a Familial Alzheimer patient (PS1 mutation) (4 FAD), Alzheimer patients (5 AD) and patients with vascular dementia (6VAD) as described in example 7
  • the level of ⁇ -amyloid(i -42) was measured as described in example 3 Data are expressed in pg/ml
  • Rab3a and SNAP25 were expressed in E. coli using a PL based expression system, pIGRHISARab3a and pIGRH6SNAP25a, respectively
  • the correct plasmid was transformed into MCI 061 pACI (Wertman et al , 1986) with a thermo sensitive cl repressor A coumassie stainable band around 25 kDa was visible on a 12 5% acrylamide gel, indicating a reasonable expression level
  • the recombinant proteins were made as a fusion protein containing 6 additional histidine residues to allow rapid purification over NiTMAC columns More than 10 mg of recombinant Rab3a and SNAP25 were purified to at least 95% homogeneity using 3 liter of heat-induced E. coli (Hochuli, 1988, Van Gelder et al , 1993)
  • a sandwich ELISA based on a monoclonal Rab3a or SNAP25 antibody as capturing antibody and a biotinylated immuno-affinity-purified polyclonal antibody as detector antibody was developed Maxisorp microtiterplates were coated with Affini Pure Goat anti-Mouse IgG (Jackson Immuno Research Laboratories, Inc , West Grove, Pennsylvania, USA, Cat No 115- 035-144) 1% BSA (Clinical Grade 98% fatty acid free, ICN, Biomedical Research Products, Costa Mesa, CA, USA, Cat No 105033, Lot No 6p384 ) in PBS was used as blocking buffer
  • Coupled peroxidase was detected via TMB, H 2 O 2 substrate solution
  • Example 2 Presence and detection of ⁇ -synuclein in cerebrospinal fluid
  • E. coli The E. coli proteins were subsequently run on a SDS-PAGE and immunoblotted with the commercial monoclonal antibody from Transduction Labs (Lexington, KY, USA) This monoclonal antibody showed to be specific for ⁇ -synuclein, mapping the carboxyterminal half of the synuclein protein (Fig 3)
  • Alpha-synuclein was purified from 3 liters of induced E. coli cultures resulting in purification of more than 10 mg ⁇ -synuclein (more than 90% pure estimated from coumassie and silverstained gels)
  • the protein was injected into mice following several immunization schemes (Table 3) After 4 injections the titer to ⁇ -synuclein was evaluated in a coating ELISA Six mice had a titer above 100000 (titer defined as serum dilution resulting in OD value twice the background)
  • spleen cells were retrieved from animal 312 (m3) and used for cell fusion mainly according the procedure as described by Kohler and Milstein (1975)
  • hybridoma's were tested for the presence of specific antibodies in a direct coating assay Subsequently, they were retested on dot-blot of E. coli lysates containing ⁇ -, ⁇ -, ⁇ - synuclein and deletion mutants from ⁇ -synuclein in order to select for hyb ⁇ domas that produce antibodies that recognize a different epitope on the synuclein protein
  • Biotinylation is performed according to well-established procedures (Bonhard et al , 1984) using D-Biotinoyl-eta-aminocaproic acid N-Hydroxysuccinimide Ester (Boehringer-Mannheim, Brussels, Belgium, Cat No 1008960)
  • a sandwich ELISA based on an ⁇ -synuclein antibody as capturing antibody and one of the biotinylated monoclonal antibodies as detector antibody is developed Maxisorp microtiterplates are coated with Affini Pure Goat anti-Mouse IgG (Jackson Immuno Research Laboratories, Inc , West Grove, Pennsylvania, USA, Cat No 115-035-144) 1% BSA (Clinical Grade 98% fatty acid free, ICN, Biomedical Research Products, Costa Mesa, CA, USA, Cat No 105033, Lot No 6p384 ) + 1% mice serum in PBS is used as blocking buffer
  • Anti- ⁇ - synuclein Transduction Labs, Lexington, KY, USA
  • recombinant antigen is added at different concentrations
  • biotinylated anti- ⁇ -synuclein monoclonal antibody is added in the presence of 1% mice antibodies at a concentration chosen for optimal background-signal ratio.
  • the biotinylated rabbit antibodies are then detected via horse-radish labeled streptavidine (Jackson Immuno Research Laboratories, Inc., West Grove,
  • Coupled peroxidase is detected via TMB, H O 2 substrate solution. Reaction is stopped after 30 min with 2N H 2 SO 4 and absorbance is measured at 450 nm.
  • Example 3 Detection of other neurological markers in cerebrospinal fluid
  • Total tau was measured with the tau antigen test, using AT 120 as capturing antibody and biotinylated HT7-BT2 as detector antibody (INNOTEST hTau antigen, Innogenetics, Gent, Belgium).
  • Monoclonal antibody AT120 reacts equally well with both normal and hyperphosphorylated human tau protein (Vandermeeren et al., 1993)
  • monoclonal antibody HT7 also reacts equally well with both normal and hyperphosphorylated human tau protein, while monoclonal antibody BT2 preferentially recognizes normal tau (Goedert et al., 1994).
  • Affinity purified tau protein prepared as described previously (Mercken et al., 1992b), was used as standard.
  • Phospho-tau was measured with a sandwich ELISA, using as HT7 as capturing antibody and biotinylated AT270 as detector antibody (INNOTEST phospho-tau(181), Innogenetics, Gent, Belgium). AT270 specifically recognizes phospho-tau (International application published under WO 95/17429).
  • ⁇ -amyloid ( ⁇ -42) concentrations were measured with the Innotest ⁇ -amyloid(i. 42) (Innogenetics, Gent, Belgium).
  • the assay is a sandwich-type ELISA, in which a first monoclonal antibody, 2 IF 12 (specific for the carboxy-terminus of amyloid), is used as capturing antibody and biotinylated 3D6 (specific for the amino-terminus), is used as detector antibody
  • 2 IF 12 a first monoclonal antibody
  • biotinylated 3D6 specific for the amino-terminus
  • the combination of 21F12/3D6 allows the specific detection of amyloid ( ⁇ - 42) peptide
  • Some cross- reactivity is observed for amyloid ( ⁇ -4 3) but not for shorter peptides (Citron et al , 1997, Johnson-Wood et al , 1997)
  • 2 IF 12 antibody was suspended in 10 mM Tris- 10 mM NaC
  • ⁇ -amyloid(i-40) concentrations were measured using a C-terminal specific affinity purified polyclonal antibody from Quality Controlled Biochemicals (QCB, Hopkinton, MA, USA) as capturing antibody and 3D6 (Citron et al , 1997, Johnson-Wood et al , 1997) as detector antibody Nunc maxisorps microtitre plates were coated for 2 hrs at 25°C with 5 ⁇ g/ml affinity purified goat anti rabbit IgG (H+L) (Jackson Immuno Research Laboratories, Inc , West Grove, Pennsylvania, USA, Cat No 111-005-144) in 10 mM Tris-10 mM NaCl buffer Thereafter, plates were blocked with PBS-0 1% casein overnight at 4°C The rabbit polyclonal (QCB, Hopkinton, MA, USA, Cat No 44-348-20) was added at a concentration of 0 5 ⁇ g/ml for 1 hr at 25°C After several wash steps, 100 ⁇ l CSF
  • Neuromodulin was also measured with a sandwich-type ELISA, using two epitope-specific monoclonal antibodies (NM2, NM4; Oestreicher et al, 1994).
  • NM2 was selected as capturing antibody
  • biotinylated NM4 as detector antibody.
  • Recombinant neuromodulin was used as standard.
  • NSE measurements were based on a sandwich ELISA using an anti-NSE monoclonal antibody, 2E7, as capturing antibody, and the peroxidase-labelled anti-NSE monoclonal antibody, 10C1, as detector antibody.
  • Purified NSE from human brain was used as standard (Vanmechelen et al., 1997).
  • Protein concentrations were determined with the BCA protein reagent (Pierce, Rockford, Illinois, USA).
  • Example 4 Combination assay, making use of CSF-Rab3a, CSF-SNAP25, CSF-tau detection of Alzheimer's disease and the differentiation of Alzheimer's disease versus age-matched controls
  • the Alzheimer's disease (AD) group included 32 patients, 15 men and 17 women, with a mean age + SD of 75.0 + 6.6 years.
  • the vascular dementia (VAD) group existed of 20 patients, 10 men and 10 women, with a mean age ⁇ SD of 81.0 + 7.0 years.
  • the control group contained 18 individuals, 7 men and 11 women, with a mean age + SD of 67.5 ⁇ 5.5 years. Diagnosis of probable AD was made by exclusion, in accordance with the NTNCDS-ADRDA criteria (McKhann et al., 1984).
  • VAD was diagnosed in patients with transitory ischemaemic attacks and/or stroke episodes in relation to the evolution of dementia and/or CT finding of large infarcts and/or multiple lacunas, and/or history of or clinical findings of severe vascular diseases, such as arterial hypertension or diabetes mellitus with complications.
  • the control group consisted of individuals without histories, symptoms or signs of psychiatric or neurological disease, malignant disease, or systemic disorders (e.g. rheumatoid arthritis, infectious disease).
  • the cognitive status was examined using the Mini-Mental state examination (Folstein et al., 1975). Individuals with scores below
  • likelihood ratios were determined (Table 5). Since Rab3a and SNAP25 levels are correlated, one can only use either Rab3a or SNAP25 in combination with tau and ⁇ -amyloid ( ⁇ -42) (Fig. 8). Thus likelihood-ratios for combinations of Rab3a and
  • Example 5 Combination assay, making use of CSF-tau, CSF-neuromodulin and CSF- neuron-specific enolase as neurological markers for the diagnosis of chemotherapy-induced neuronal damage in children treated for leukemia
  • Burkitt's lymphoma and 2 patients had anaplastic large cell lymphoma (ALCL). Eight of these patients were studied longitudinally. A second and largest group consisted of 42 patients with non-B-cell acute lymphoblastic leukemia/non Hodgkin's lymphoma (NB ALL/NHL), treated according to the 'European Organization for Research and Treatment of Cancer' (EORTC) protocol 58881 (Table 7b). In this second group, 18 children had CD10(+) blasts or common NB
  • M0, and 1 patient each with Ml, M2 or M7 phenotype were treated according to the EORTC 58921 protocol (Table 7c) and followed longitudinally.
  • This group includes 5 children with medulloblastoma (3 staging, 1 during treatment, 1 follow up),
  • Lumbar punctures were performed for routine analysis either at baseline for diagnostic work-up or just prior to the IT administration of chemotherapy.
  • Five ml of CSF was collected in different polypropylene tubes. One sample was centrifuged immediately at 1500 rpm for 2 minutes to eliminate cells and other insoluble material. The supernatant was stored at -70°C for subsequent analysis. The number of freeze/thaw cycles was restricted to a minimum. Routine CSF measurement included cytology, protein concentration, glucose, etc.
  • Tau levels at diagnosis were analyzed for each subgroup of patients (Fig. 9). Tau levels at diagnosis ranged from 66 to 1500 pg/ml.
  • the data for tau, neuromodulin, ⁇ -amyloid ( ⁇ . 2) , ⁇ - amyloid(i - 0) , serum LDH and CSF white blood cell count (Fig. 10) show no obvious difference in patient groups with and without Down syndrome or between diagnositc groups.
  • no correlation was found between the tau level and WBC or LDH levels. No significant correlation was found between the tau concentration and the age of the children.
  • tau levels at diagnosis did not correlate with tumor burden, as reflected by the white blood cell count
  • Figure 14a shows the evolution of tau in individual patients with AML.
  • Patients 11, 12 and 23 did not have a significant increase in tau during treatment (Table 7c).
  • Patient 12 from whom LPs were taken at days 1 and 8, had an aggressive disease and died early after bone marrow transplantation. From one out of two patients with Down's syndrome, longitudinal data were available, and this patient had tau levels raising slightly above 300 pg/ml, in contrast to the evolution of tau levels in the CSF of patient 11 and 23.
  • Patient 69 with evidence of CNS invasion at diagnosis, had a tremendous increase of tau and neuromodulin (Fig. 14b) in the CSF. This child is still under treatment, and is in complete remission at the moment.
  • Example 6 Combination assay for the diagnosis of brain damage resulting from perinatal asphyxia
  • Perinatal asphyxia may be associated with neuronal damage
  • CSF neurological markers may complement clinical data in the evaluation of hypoxic-ischemic events (Garcia- Alix, 1994) It has become increasingly evident that modified brain metabolic activity is reflected by changes in components in the CSF The perinatal levels of the CSF markers and the distribution of changes due to aspyxia are evaluated
  • Example 7 Combination assay, making use of CSF-neuromodulin.
  • CSF-tau and CSF- ⁇ -amyloid man as neurological markers for the specific detection of Alzheimer's disease and for the differentiation of Alzheimer's disease versus control subjects
  • AD Alzheimer's disease
  • VAD vascular dementia
  • CSF-tau and CSF- ⁇ -amyloid (1 . 4 2) were significantly altered in the AD patients
  • CSF- neuromodulin was significantly increased in AD
  • CSF-tau level was significantly raised compared to the control subjects
  • Three of the 7 patients with memory- impairment had CSF-tau levels above the maximum defined by the control group (280 pg/ml) Due to improper storage, CSF- ⁇ -amyloid ( ⁇ -42 ) levels could only be determined in 3 patients with memory impairment
  • levels below 300 pg/ml were found
  • One of these patients had also an elevated CSF-tau level
  • the other patient had a CSF-tau level of 278 pg/ml, i e just below the maximum defined by the control group
  • an increase of CSF-tau level of 327 pg/ml was observed, together with a ⁇ -amyloid
  • Example 8 Combination assay, making use of CSF-neuromodulin.
  • CSF-beta- amyloid ⁇ .47 and CSF-tau as neurological markers for the specific detection of Alzheimer's disease, to differentiate Alzheimer's disease versus control subjects, for the specific detection of Parkinson disease, to differentiate Parkinson disease versus control subjects and to differentiate between Alzheimer's disease and Parkinson disease.
  • CSF was obtained from 60 patients with Alzheimer's disease, 23 patients with Parkinson disease and 32 age-mached controls.
  • ⁇ -amyloid ( ⁇ - 42 ) and neuromodulin were selected to differentiate Alzheimer's disease patients from Parkinson disease patients with a sensitivity of 94.8% (Cl 85.6%-98.9%) for the Alzheimer's disease patients and a specificity of 78.3% (Cl 56.3%-92.5%) for the Parkinson disease patients. Further analysis of a larger number of samples will enable to demonstrate a statistically significant improvement of the differentiation between Alzheimer's disease and Parkinson disease by use of these three markers.
  • Example 9 Use of CSF- ⁇ -synuclein as a neurological marker to differentiate between Lewv Body dementia and Alzheimer's disease
  • CSF is obtained from 30-40 patients with Alzheimer's disease, 10-20 patients with Lewy Body dementia and 10-20 age-mached controls. 9.2 Assay for the differential diagnosis of Alzheimer's disease and Lewy Body dementia
  • the concentration of ⁇ -synuclein in the CSF of the different patients and control groups is quantified.
  • a significant different mean value for the ⁇ -synuclein level in the Alzheimer's disease group versus the mean value for the ⁇ -synuclein level in the Lewy Body dementia group allows us to differentiate between both types of dementia. Differentiation between Alzheimer's disease and Lewy Body dementia is further improved by combining the quantification of ⁇ -synuclein with the quantification of at least 2 other neurological markers (such as tau and ⁇ -amyloid ( ⁇ . 42 ).
  • Example 10 Combination assay, making use of CSF-beta-amyloid ⁇ -m.
  • CSF-tau and CSF-phospho-tau as neurological markers for the specific detection of Frontal Temporal Lobe dementia and to differentiate Frontal Temporal Lobe dementia versus other dementia
  • CSF is obtained from 30-40 patients with Frontal Temporal Lobe dementia and 10-20 age- mached controls.
  • the level of ⁇ -amyloid (1 _42), tau and phospho-tau in the CSF of the different patients with Frontal Temporal Lobe dementia and in the control patients is quantified.
  • the mean values for the level of tau and phospho-tau in the patients with Frontal Temporal Lobe dementia is significantly increased compared to mean values for the level of tau and phospho-tau in the control patients.
  • the patients with Frontal Temporal Lobe dementia show a decreased level of ⁇ -amyloid(i. 2 ) compared to the control patients.
  • Example 11 Combination assay, making use of CSF-beta-amyloid ⁇ -47 . CSF-tau and detection of vascular problems in Alzheimer's disease and to differentiate vascular disease from other forms of Alzheimer's disease
  • CSF and plasma is obtained from 30-40 patients with vascular disease, from 30-40 patients with other forms of Alzheimer's disease and from 10-20 age-mached controls.
  • the level of tau and ⁇ -amyloid ( ⁇ .4 2 ) in the CSF and the level of ⁇ -amyloid(i- 2 ) in the plasma of the different patients with vascular disease, with other forms of Alzheimer's disease and in the control patients is quantified.
  • a quantitatively different level of plasma- ⁇ -amyloid ( ⁇ -42) , CSF-tau and CSF- ⁇ -amyloid(i- 2) enables the differentiation between the vascular disease patients and the patients with other forms of Alzheimer's disease.
  • Citron M Westaway D, Xia W, Carlson G, Diehl T, Levesque G, Johnson- Wood K, Lee M,
  • Bancher C Cras P, Wiltfang J, Mehta PD, Iqbal K, Pottel H, Vanmechelen E,

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EP99934543A 1998-07-03 1999-06-29 Differentielle diagnose von neurodegeneration Expired - Lifetime EP1095278B1 (de)

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EP99870069 1999-04-09
EP99870069 1999-04-09
EP99934543A EP1095278B1 (de) 1998-07-03 1999-06-29 Differentielle diagnose von neurodegeneration
PCT/EP1999/004483 WO2000002053A2 (en) 1998-07-03 1999-06-29 Differential diagnosis of neurodegeneration

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EP1095278B1 (de) 2005-12-07
DE69928809D1 (de) 2006-01-12
AU5029099A (en) 2000-01-24
JP4580455B2 (ja) 2010-11-10
JP2002519702A (ja) 2002-07-02
ATE312349T1 (de) 2005-12-15
BR9911291A (pt) 2001-12-04
CA2329523A1 (en) 2000-01-13
DE69928809T2 (de) 2006-08-31
JP2010019864A (ja) 2010-01-28
WO2000002053A3 (en) 2000-02-24
ES2255280T3 (es) 2006-06-16
WO2000002053A2 (en) 2000-01-13
CN1316055A (zh) 2001-10-03
JP2006091025A (ja) 2006-04-06
AU754062B2 (en) 2002-10-31
DK1095278T3 (da) 2006-04-18
HK1036834A1 (en) 2002-01-18

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